1. Molecular Cellular Biology-I (MCB-I) PCB 6025 M. Alejandro Barbieri M. Alejandro Barbieri Office: HLS 318C/214 Hours: by appointment. Email: barbieri@fiu.edu barbieri@fiu.edu

2. 1-Regulatory RNA, 2-RNA interference and micro RNA, 3-Retroviruses, 4-Transposons and Retroposons, 5-Promoters and Enhancers, 6-Activating Transcription, 7-RNA Splicing and Processing, 8-Chromosomes-Nucleosomes, 9-Controlling Chromatin Remodeling and Structure. TOPICS-I

3. 10-Gene Regulation I, 11-Gene Regulation II, 12-Protein Synthesis TOPICS-II

4. 4 Books Molecular Biology of the Cell Genes IX/X/... Any....

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6. Presentation: Topic selection/student Regulatory RNA (review and paper) RNA interference Micro RNA Transposons, Retroposons and Retroviruses Promoters and Enhancers Activating Transcription RNA Splicing and Processing Controlling Chromatin Structure and Chromatin remodeling Retroviruses Gene Regulation

7. 7 Evaluation: 1- Exam= 50% (Parts-I and II) 2-Presentation 1=50%* (review and specific paper, idea, concept and experimental-selected by the instructor) 2a-presentation: 40% 2b-participation: 10% (*)-Each student will make a presentation for 15 min. (for specific papers) and 20 min (for review papers) with discussion (2-3 questions).

8. Exam (Topics plus selected papers) Open/Close book

9. Regulatory RNA

10. 10 MicroRNAs Are Regulators in Many Eukaryotes Animal and plant genomes code for many short ( ∼ 22 base) RNA molecules called microRNAs. MicroRNAs regulate gene expression by base pairing with complementary sequences in target mRNAs. C. elegans: regulator gene lin4 and its target gene lin14 (lin: Proteins for larval development)

11. 11 RNA Interference Is Related to Gene Silencing RNA interference triggers degradation of mRNAs complementary to either strand of a short dsRNA. Figure 13.21

12. 12 dsRNA may cause silencing of host genes. Figure 13.22

13. What is RNA interference? Shooting down mRNA

14. Background Background What is it? What is it? Why use it? Why use it? The mechanism and process The mechanism and process Experimental considerations Experimental considerations RNAi

15. 15 Plasmid Virus

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17. 17 Jorgensen 1990 van der Krol 1990 Gene injection (pigmentation Enzyme-petunias) Expectation: more red color Co-suppression of transgene and endogenous gene. Bill Douherty and Lindbo 1993 Gene injection with a complete tobacco etch virus particle. Expectation: virus expression Co-suppression of transgene and virus particles via RNA. Hamilton and Baulcombe 1998 Identification of short antisense RNA sequences dsRNA? How? Fire and Mello 1998 Injection of dsRNA into C. elegans RNA interference (RNAi) or silencing Ambros 1993 (2000) Identification of small RNA in C. elegans (micro RNA)

18. 18 Shooting mRNA means RNA interference

19. What is RNA interference? --Gene “knockdown” --A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus. Why? --A way for the cell to defend itself.

20. Why use RNAi? 1. The most powerful way to inhibit gene expression and acquire info about the gene’s function fast 2. Works in any cell/organism 3. Uses conserved endogenous machinery 4. Potent at low concentrations 5. Highly specific.

21. The mechanism of small interfering RNAs (siRNAs) What happens? dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous RNA.

22. 22 RNA interference: --A type of gene regulation --Involve small RNA molecules --Induce a double stranded RNA The RNAi process

23. 23 Step 1 dsRNA is processed into sense and antisense RNAs 21-25 nucleotides in length have 2-3 nt 3’ overhanging ends Done by Dicer (an RNase III-type enzyme)

24. 24 Step 2 The siRNAs associate with RISC (RNA- induced silencing complex) and unwind

25. 25 Step 3 the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs.

26. 26 Step 4 RISC cuts the mRNA approximately in the middle of the region paired with the siRNA The mRNA is degraded further

28. 28 Catalysis: RdRP copies RNA making more ds RNA. Dicer complex: RNAase III with ATP hydrolysis requirement. Dicer cuts, unwinds dsRNA and generates more siRNA. More RdRP is activated and more dsRNA is made. RISC complex:RNA-Inducible Silencing Complex with ATP hydrolysis. ssRNA (exogenous) RNA-dependent RNA polymerase (endogenous) (RdRP)

29. 29 Gene regulation by small RNAs Small temporal (St) RNAs prevent translation to stop gene expression quickly siRNAs degrade mRNA to stop gene expression quickly Dicer gene in C. elegans

30. 30 --MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression (down-regulation). --miRNAs are encoded by genes that are non-coding RNAs ( no proteins are made) --Stem-loop or hairpin loop intra-molecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. -your RNAi?

31. 31

32. Experimental Considerations Transfection method: 1-Lipofectamine 2000--cationic lipids to bind siRNA and neutral lipids to allow escape from Endosomes 2-Plamids/Viruses--express small fragment of hairpin DNA Transfection efficiency Negative controls --scrambled siRNA Off-target effects: Sense (or antisense) strand is homologous to another sequence Activation of stress response pathways “apoptosis”

33. 33 http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html

34. Growth Factor Receptor Binding Protein (Grb) 2-mediated Recruitment of the RING Domain of Cbl to the Epidermal Growth Factor Receptor (EGFR) Is Essential and Sufficient to Support Receptor Endocytosis Fangtian Huang and Alexander Sorkin

35. 35

36. 36 Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, we have generated cell lines in which endogenous Grb2 was replaced by a modified yellow fluorescent protein (YFP)-tagged Grb2 and it was expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis.

37. 37 To generate HeLa cells stably expressing Grb2-YFP, endogenous Grb2 was knocked down using vector-based short hairpin RNA (shRNA) with simultaneous expression of Grb2-YFP that has silencing mutations rendering this construct insensitive to shRNA.

38. 38 pSilencer1.0-U6 vector-- pSuper-H1 vector-- Type III RNA pol III promoter ----U6 small nuclear promoter (U6) ----human H1 promoter (H1) retrovirus//inducible